PROJECT SUMMARY Histone modifications are key constituents of the epigenome regulating growth and development, and they control nuclear processes including transcription. Misregulation of histone modifications is implicated in the pathogenesis of many human diseases. Numerous genome-wide studies have clearly shown that multiple histone modifications co-occupy genomic regions and define chromatin sub-domains. Chromatin modifying or remodeling complexes contain subunits with specialized interaction domains that bind different histone modifications. Therefore, combinatorial histone modifications on a nucleosome might play a key role in regulating transcription, but this aspect is very poorly understood. Apart from nucleosomes asymmetrically modified with H3K4me3-H3K27me3 marks implicated in transcriptional regulation at poised or developmentally regulated genes, other combinatorial modifications or asymmetrically modified nucleosomes remain unknown. We have identified a novel H3K4me3-H3K4acetyl (ac) dually modified mononucleosomes in yeast as well as human cells. Using ReChIP-seq, we have determined that nucleosomes with the H3K4me3-H3K4ac bivalent mark are present at actively transcribed genes and enriched around transcription start sites both in yeast and human genomes. H3K4me3 is often used as a surrogate for gene expression, but this mark is also found at inactive or poised genes. Therefore, we hypothesize that nucleosomes dually modified with H3K4me3-H3K4ac marks are the best indicators of active transcription and transcription start sites in genomics studies. In Aim 1, we will further test our hypothesis by mapping the genomic location of H3K4me3-H3K4ac bivalent mark in other model eukaryotes (Drosophila and C. elegans). Using ReChIP-seq, we will map the genome-wide occupancy of H3K4me3-H3K4ac dual mark in undifferentiated and lineage-committed human embryonic stem cells and confirm its active establishment at transcribed genes. The orientation of H3K4me3 and H3K4ac marks within a nucleosome might constitute a further epigenetic regulatory signal during transcription. Using the high resolution ChIP-nexus approach, we have determined a distinct orientation for H3K4ac and H3K4me3 marks within yeast nucleosomes. In Aim 2, we will determine whether H3K4me3 and H3K4ac marks also acquire specific orientation in human cells. Using biochemical and genomic approaches, we will aim to gain mechanistic insights into the functions of H3K4ac-H3K4me3 bivalent mark as a recruiter and/or regulator of chromatin modifiers and remodelers during transcription. Successful completion of our proposed studies will provide a paradigm-shifting concept and establish the novel H3K4me3-H3K4ac bivalent modification as a genuine mark of active transcription. Our study will be the first to provide a comprehensive epigenomic map for a novel histone bivalent mark functionally connected to transcriptional activity in multiple eukaryotes and during growth and differentiation in human cells. Importantly, our study will usher in the next wave of epigenomics studies aimed towards identifying and investigating the functions of novel combinatorial histone marks.